1. Field of the Invention
The present invention relates to analytical separation of samples into components in capillaries, and more particularly to the detection of the separated components which have been labeled by radioisotopes.
2. Description of Related Art
Analytical separation of components of a sample has widely been carried out using capillaries as separation channels. For example, capillary electrophoresis has proven useful as a highly efficient method for analytical separation of a minute amount of sample. In this process, an electric field is applied between the two ends of a capillary tube into which a buffer or electrolyte containing the sample is introduced. The electric field causes the components of the sample to migrate through the tube. The components will have different electrophoretic mobilities so that the components are resolved into zones or bands in the separation channel during migration through the channel.
Early in the development of capillary electrophoresis, it was noted that the successful detection of separated sample components present within the narrow confines of the capillary separation channel posed a major challenge. In response to this challenge, much research has been directed toward the development of sensitive and selective detectors for capillary electrophoresis. To improve selectivity of detection, it is desirable to employ detectors which respond only to certain sample components but not to others, thus permitting detection of the origin of certain sample components despite chemical changes and the presence of other components and substances. Since the amounts of materials used in capillary electrophoresis are so minute, detectors used must have high sensitivity. One way to increase detection selectivity and sensitivity is to label the sample components with one or more radioisotopes.
While state-of-the-art radiation detection technology offers extremely high sensitivity and selectivity in detection of sample components in the capillary separation channel, there are certain limitations. In the past, detection of radioisotope-labeled components within the capillary separation channel has been accomplished during electrophoretic separation. A radiation detector is placed along the capillary tube at a fixed location. The labeled components move past the detector at a finite velocity. This technique is referred to as "flow counting detection". The ability to accurately detect and quantify the components is dependent on the residence time of the components within the detection range of the detector and the zone broadening which occurs due to diffusion of the components in the separation channel. The residence time affects sensitivity and diffusion affects both resolution and sensitivity. While detection sensitivity can be improved by increasing the residence time of the components (for example, by reducing the applied electric potential to slow down or stop the flow in the separation channel), zone broadening caused by diffusion is more pronounced as residence time is increased thereby degrading the resolution. Hence, there is a trade-off between sensitivity and resolution in the flow counting approach of detection.
Sample zones are caused to broaden by at least two mechanisms. First, the applied electric field causes resistive Joule heating of the buffer to create a temperature gradient between the axis of the separation channel and the walls of the channel. This creates a convective recirculation within the channel thereby causing the sample zones to broaden. Second, there is a concentration gradient between the zones and the adjacent buffer which causes diffusion across the zone boundaries. Such diffusion is more pronounced at higher temperature.
While broadening of sample zones causes by convection can be reduced to some extent but not eliminated by effective heat dissipation through the surrounding capillary walls, longitudinal diffusion of sample components due to concentration differential is still present.